Effect of integrated crop-livestock systems in carcass and meat quality of Nellore cattle

Accepted Manuscript

Effect of integrated crop-livestock systems in carcass and meat quality of Nellore cattle Patr´ıcia Aparecida Cardoso da Luz , Cristiana Andrighetto , Gelci Carlos Lupatini , Helena Sampaio Aranha , Gustavo Antunes Trivelin , Gustavo Pavan Mateus , Carolina Toledo Santos , Caroline de Lima Francisco , Andre´ Michel Castilhos , Andre´ Mendes Jorge PII: DOI: Reference:

S1871-1413(18)30743-1 https://doi.org/10.1016/j.livsci.2018.11.018 LIVSCI 3581

To appear in:

Livestock Science

Received date: Revised date: Accepted date:

17 July 2018 27 November 2018 28 November 2018

Please cite this article as: Patr´ıcia Aparecida Cardoso da Luz , Cristiana Andrighetto , Gelci Carlos Lupatini , Helena Sampaio Aranha , Gustavo Antunes Trivelin , Gustavo Pavan Mateus , Carolina Toledo Santos , Caroline de Lima Francisco , Andre´ Michel Castilhos , Andre´ Mendes Jorge , Effect of integrated crop-livestock systems in carcass and meat quality of Nellore cattle, Livestock Science (2018), doi: https://doi.org/10.1016/j.livsci.2018.11.018

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ACCEPTED MANUSCRIPT Highlights The carcass characteristics of Nellore cattle are not influenced by the different ICLS.

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Trees present in the pasture does not interfere in the physical composition of the meat.

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The microbiology of the meat was not influenced by the different ICLS.

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ICLS with and without the presence of trees can be recommended for Nellore cattle.

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ACCEPTED MANUSCRIPT Effect of integrated crop-livestock systems in carcass and meat quality of Nellore cattle

Patrícia Aparecida Cardoso da Luz, Cristiana Andrighetto, Gelci Carlos Lupatini, Helena Sampaio Aranha, Gustavo Antunes Trivelin, Gustavo Pavan Mateus, Carolina Toledo Santos, Caroline de Lima Francisco, André Michel Castilhos, André Mendes Jorge

P.A.C. Luz, C.L. Francisco, A.M. Castilhos and A.M. Jorge. São Paulo State University

Botucatu 18618-000, SP, Brazil.

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(UNESP), College of Veterinary Medicine and Animal Science, Department of Animal Science,

C. Andrighetto, G.C. Lupatini, H.S. Aranha and G.A. Trivelin. UNESP, College of Agrarian and Technological Sciences, Dracena, SP 17900-000, Brazil.

G.P. Mateus. APTA, Paulista Agency for Agribusiness Technology, Polo Regional Extreme West -

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Andradina, SP 16900-970, Brazil.

C.T. Santos. UNESP, College of Agricultural Science, Department of Economy, Sociology and Technology, Botucatu, 18.610-307, SP, Brazil.

Corresponding author: Patrícia Aparecida Cardoso da Luz [email protected]

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*These authors contributed equally to this work.

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Abbreviations: ICLS. Integrated Crop-livestock Systems; ICL. Integrated Crop-livestock; ICLF. Integrated Crop-Livestock-Forest; PUFA. Polyunsaturated Fatty Acids; MUFA. Monounsaturated Fatty Acids; SUFA. Saturated Fatty Acids; HH. Hypocholesterolemic and Hypercholesterolemic;

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AI. Atherogenicity Index; TI. Thrombogenicity Index.

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ACCEPTED MANUSCRIPT Effect of integrated crop-livestock systems in carcass and meat quality of Nellore cattle

Abstract

Integrated crop-livestock systems (ICLS) are strategy to increase productivity and income for the farmers while simultaneously achieving sustainability. However, its effects on the carcass and meat quality of the animals produced in it need to be elucidated. Thus, the objective of this study was to

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evaluate carcass and meat characteristics of Nellore cattle finished in ICLS without shade availability (integrated crop-livestock: ICL) and with two tree densities (integrated crop-livestockforest, ICLF, at 196 trees/ha and 448 trees/ha). The experimental design was in complete blocks, with three treatments (ICL, ICLF-1L and ICLF-3L) and four replicates per treatment, totaling 12 experimental plots. Sixty castrated Nellore cattle of approximately 28±2.81 months of age and

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mean weight at 385.71±27.17 kg. After the slaughter of the animals, 48 samples of the longissimus thoracis muscle were collected for carcass and meat quality analyzes. There were no differences (P>0.05) between the treatments for the average daily gain and final live weight, contributing to the absence of difference in weight and yield of the hot carcass, forequarter, hindquarter and flank (P>0.05). Similarly, no were found difference (P>0.05) between treatments for initial and final

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ribeye area, initial and final backfat thickness, marbling, pH and glycogen, both measured at 2 h and 24 h post mortem. The proximate composition, cholesterol, pH, cooking loss, shear force, collagen,

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meat color and microbiological analysis presented no differences (P>0.05) among the evaluated treatments. However, there was a trend of a lower amount of C17:1n9 and C20:4n6 polyunsaturated fatty acids (P = 0.05 and P = 0.06, respectively), but this result did not influence the amount of

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PUFA and the ω6:ω3 ratio (P>0.05), which was lower in all treatments with the recommended to prevent cardiovascular problems. In addition, although the thrombogenicity index (IT) presented a

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tendency of higher value in the meat of the animals in ILPF-3L systems (P=0.08), all treatments presented higher level than recommended in the diet for good health. Thus, concludes that the

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integration systems that use the tree component do not benefit the quality of the final product, the meat of Nellore cattle, but should be recommended because they do not interfere in quality of the same.

Keywords: integrated crop-livestock-forest, longissimus thoracis, muscle glycogen, ribeye area, tenderness.

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1. Introduction According to estimates by the FAO (2016), approximately 15% of the world's population is malnourished and, for the coming decades, there is an estimated population increase from roughly 6.5 billion today to 9.2 billion by 2050, which may be accompanied by global hunger, as more than 1 billion of this increase will occur in Africa. As a result, agricultural production and productivity growth remain essential to guarantee better nutrition (FAO, 2013).

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Thus, there is a challenge of expanding and improving food, fiber and energy production, especially for the coming decades, with the need to establish systems with low environmental impact and resistance to climate change (Rockstrom et al., 2009; Godfray et al. al., 2010).

In this context, integrated crop-livestock systems may be one of the best alternatives for demand-driven increase of the expansion of global food production for the future (Franzluebbers

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and Stuedemann, 2007; Wirsenius et al., 2010), without occupying new areas, together with the urgent need for sustainable agriculture and livestock breeding.

Among the systems, the Integrated Crop-Livestock (ICL) is considered as the "new green revolution in the tropics" (Mateus et al., 2012). And, more recently, with the introduction of the arboreal component - Integrated Crop-Livestock-Forest (ICLF), it constitutes a new paradigm in

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Brazilian agriculture, capable of benefiting soil and forage, apart from providing favorable microclimate, increasing the thermal comfort index for the animals kept in the shade of the trees

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(Silva et al., 2011).

This technological advance is mainly due to the fact that most pasture areas, especially in Central Brazil, are used under climatic conditions that favor caloric stress of medium to severe

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types in the animal (Porfírio da Silva, 2003). The impact of heat-derived stress changes the quality of the meat of the animals submitted to

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it during long periods (Gregory, 2010), with possible effects on muscle metabolism that are directly related to a drop in pH during slaughter and, consequently, color and tenderness of the meat, which

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becomes dark, since high pH causes the enzymes to use oxygen rapidly, reducing the proportion of oxygenated red pigments (oxymyoglobin); as well as firm, resulting from the lower activation of the calpain enzyme at high pH; and dry, due to the higher water retention capacity, because pH is far from the isoelectric point of muscle proteins (Guàrdia et al., 2005; Maganhini et al., 2007). In addition, there is a greater development of deteriorating microorganisms in the meat at higher pH values (Guàrdia et al., 2005). The introduction of trees in pastures, besides promoting well-being of the animals, can also increase the quality of the forage available to them (Sánchez, 2001). And, since the meat of grassfed ruminants contains a higher proportion of healthy lipids and antioxidants that are important for 4

ACCEPTED MANUSCRIPT human health (Wood et al., 1999), it is necessary to evaluate the fatty acid profile of the beef from bovines submitted to different models, since such data are scarce in the available literature. Thus, given the hypothesis that the ICLF can promote well-being of the animals and improve the quality of the fodder, resulting in higher quality of the meat, the present study was performed with the objective of evaluating the characteristics of the carcass and meat of bovines of the Nellore breed, slaughtered within integrated agricultural production systems, both with no available shade

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(ICL) and at two different tree density values (196 trees/ha: ICLF-1L and 448 trees/ha: ICLF-3L).

2. Materials and methods

The experiment was carried out in accordance with the ethical principles for animal tests (Protocol No. 101/2014 - CEUA) determined by the Ethics Committee on Animal Use (CEUA) of

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the Faculty of Veterinary Medicine and Animal Science - UNESP, Câmpus de Botucatu, Brazil.

2.1. The experimental site and the climatic conditions

The experiment was carried out at the Paulista Agency for Agribusiness Technology (APTA), Far West Regional Pole, located in the municipality of Andradina (20° 53' 38'' south latitude, 51° 23' 1'' west longitude and an altitude of 400 m), west of the São Paulo state.

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The predominant climate in the region is Aw, being characterized as a highland tropical climate, with hot and rainy summer and dry winter, according to the Köppen climate classification

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system (Alvares et al., 2013). According to the agency's own weather data, the region's annual precipitation is around 1,257 mm, with 78% of rainfall occurring in October-April and 22% in MaySeptember, thus characterizing the dry season. Historically (1956-2013), the average annual

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maximum and minimum temperatures were 30.7ºC and 17.1ºC, respectively, with average annual precipitation of 1,181.6 mm (Unicamp, 2013). Climatic and thermal comfort data were measured

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during the experimental period (Table 1).

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2.2. Area history and experimental period The experiment was conceived in the first half of 2012, when specific treatments were

selected, as well as the division of pickets were carried out. The soil of the experimental area was classified as Dystrophic Yellow Red Latosol (Embrapa, 2013) with sandy surface layer and mean terrain slope of 6%. In July 2012, the type of the area’s soil was corrected based on the chemical analyzes (0-20 cm), which revealed the following attributes: pH (CaCl2) 4.8; M.O. 16 g dm-3; P (resin) 3 mg dm-3; K+, Ca2+, Mg2+ and H+Al 1.9; 7; 5 and 20 mmolc dm-3, respectively, S-SO42- 1 mg dm-3 and V% (base saturation) of 42%. The clay, silt and sand contents were 107; 113 and 780 g kg-1, respectively. Dolomitic limestone (PRNT 80%) and gypsum were applied and incorporated 5

ACCEPTED MANUSCRIPT into the soil, as recommended by Bulletin 100 (van Raij et al., 1997) for the São Paulo state. When preparing the soil, terracing, plowing, plowing and leveling were performed. The trees were established from November 2012 to March 2013 through manual planting of the seedlings, following the level curves present in the area (Porfírio da Silva et al., 2010). The eucalyptus clone used in the planting was the I-224 of Eucalyptus urograndis, oriented towards cellulose production, which is the commercial characteristic of the plantation region. In the planting fertilization, 350 kg ha-1 of the 04-30-16 formula were used, with the amount of 210 g per seedling (8.4 g N, 63 g P2O5, 33.6 g of K2O) for each planting pit. During the cover fertilization phase

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carried out in February 2013, 37 kg ha-1 of nitrogen, 3 kg ha-1 of zinc and 2 kg ha-1 of boron were used, applying 50 g of urea (23 g N), 9 g of zinc sulfate (1.8 g Zn) and 12 g borogran (1.2 g B) in the form of a drown under each eucalyptus seedling. In January 2014, another cover fertilization round was carried out with 123 kg ha-1 of N and using 160 g of urea (73.6 g N) in the form of a

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crown under each seedling.

The seeding of soybeans (cultivar BMX Power) was performed in December 2012 in all systems (ICL, ICLF-1L and ICLF-3L), totaling 400,000 seeds per ha-1. The mineral fertilization seedlings corresponded to the application of 12 kg ha-1 of N, 90 kg ha-1 of P2O5 and 48 kg ha-1 of K2O. The cover fertilization was carried out 40 days after planting, applying 200 kg ha-1 of the

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formulated 00-20-20. The control of soybean weeds in post-emergence phase was carried out on 01/24/2013, applying herbicide based on Glyphosate (Zapp QI 620) in the dose of 1,240 g i.a. ha-1.

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In this application, cobalt-molybdenum-based fertilizer (COMO Platinum) was used for the tank mix in the dose of 150 ml ha-1 of the commercial product. The soybean harvest was carried out in May 2013, yielding an average yield of 35 sc ha-1. After the soybean harvest, weed control was

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performed in the area. The area was desiccated using Glyphosate-based herbicide (Roundup WG) at a dose of 1440 g a.i. ha-1, with a total applied volume of 250 l ha-1.

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In December 2013 the grass was sown, using Urochloa brizantha (Syn. Brachiaria brizantha) cv. Marandu, in the amount of 8.0 kg ha-1 of pure and viable seeds, planted with spacing

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between rows of 0.20 m. In the corn plantation, a 0.80 m spacing was used between lines, aiming to reach the population density of 62,500 plants per hectare, while the fertilization of seedlings corresponded to 24.8 kg ha-1 of N, 86.8 kg ha-1 of P2O5 and 49.6 kg ha-1 of K2O. 20 days after the emergence of maize plants, the cover fertilization was performed, using 92 kg ha-1 of nitrogen. The corn was harvested in April 2014, and fences were built in this period, then the drinking fountains were installed and the area remained untouched until the entrance of the animals. Between December 08, 2014 and January 9, 2015, the forage was standardized by means of mechanical weeding at a height of 15 cm, followed by nitrogen fertilization of 40 kg ha-1 of N in the form of urea. 6

ACCEPTED MANUSCRIPT In January 2015 the adaptation phase began (07/01/2015 to 11/02/2015), followed by the beginning of the rearing of the animals (beginning on 11/02/2015 and ending on 01/13/2016), with the live weight and initial age at that stage at 235.43 ± 25.46 kg and 16 ± 2.81 months, respectively, and final weight of 385.71 ± 27.17 kg and 28 ± 2.81 months of age. After the rearing phase, the finishing phase came (beginning on 01/13/2016 and ending on 01/07/2016), which makes part of this study.

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2.3. Animals, treatments, grazing management and supplementation Sixty castrated animals of the Nellore breed with 28 ± 2.81 months of age and 385.71 ± 27.17 kg of PV were used at the beginning of the finishing phase. These animals were immobilized using weight bands inside 12 pickets containing the treatments (Table 2).

The adopted grazing method was continuous stocking with variable stocking rate, using the

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put and take technique (Mott and Lucas, 1952). In each picket, five animal testers and a variable number of regulators were used, according to the need of adjusting the stocking rate to maintain the handling goal, with a mean grass height of 30 cm, which is within the range (20 to 40 cm) that is considered to be ideal pasture conditions (Silva, 2004), presenting 29±1.62 cm, 29±1.30 cm and 28±1.44 cm for ICL, ICLF-1L and ICLF-3L, respectively. The monitoring of the height conditions

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of the forage canopy was carried out by means of a ruler with a gradation in centimeters (cm), measuring the distance between the curvature of the highest leaf at the sampling point and the soil

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(Hodgson, 1990), in zigzag trajectory at 14-day mean intervals, with an average sample number of 100 points per experimental unit. The average production conditions and the chemical composition of the pasture during the experimental period are described in Table 3.

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In the dry period, which lasts from April to June, a concentrate supplement was offered for the animals with consumption of 0.7% of live weight, formulated using NRC (2001), based on the

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Mechanistic model (MLS) (Table 4). Supplements were fed daily, between 11h and 13h, trying not

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to change the behavior and grazing cycles of the animals.

2.4. The variables analyzed in the carcass After reaching the slaughter weight, the animals were weighed using a digital electronic

weighing scale (VF-B) (Valfran, São Paulo, Brazil), which has a precision of 1 kg, before being submitted to 16-hour solids fasting. The real-time ultrasonography technique was used at the beginning of the experimental period as a tool for the prediction of the ribeye area and the thickness of the subcutaneous fat thickness obtained at the height of the 12th rib. To obtain the images, ALOKA 500V equipment was used, with the linear probe of 17.2 cm and 3.5MHz, as well as coupled acoustic guide for better 7

ACCEPTED MANUSCRIPT adaptation to the anatomy of the body of the animal. The images were inserted in a portable microcomputer and analyzed using Image J (National Institute of Health, Bethesda, MD, USA). At the end of the established stay period, the animals were weighed, presenting a mean of 453.68±29.69 kg of PV and 34±2.81 months of age, and later slaughtered in a commercial slaughterhouse located in the municipality of Bariri/SP, under the guidance of State Inspection Service (SISP), at an approximately 370 km distance from the experimental site. During the slaughter operation it was possible to identify the carcasses with numbered

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labels, fixed on the right and left side. After the slaughter, the weight of the half-carcasses was recorded, which were cooled by 24 hours.

A total of 48 longissimus thoracis (LT) muscle samples were collected between the 8th and 13th ribs, which were transported to the Laboratory of Bromatology of the Faculty of Agrarian and Technological Sciences - UNESP, Dracena Campus, where were sectioned with 2.5 cm of width

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each using a band-saw (Model 255, Beccaro, Toronto, ON, Canada). After that, each cut was vacuum packed (18 μ) in a JETVAC® (200-B, Selovac, SP, Brazil) packer and frozen (-20ºC) for up to 30 days for future analysis.

The ribeye area and subcutaneous fat thickness was measured from piece photographed and analyzed using the Image J software (National Institutes of Health, Maryland, USA). The marbling

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score, was analyzed according to the methodology described by the USDA Quality Grade (1997) was used, with scores varying from 1 to 10 (1 = practically absent, 2 = traces, 3 = slight, 4 = little, 5

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= modest, 6 = moderate, 7 = slightly abundant, 8 = moderately abundant, 9 = abundant and 10 = very abundant).

During cooling of the carcasses (2ºC), the pH using a Testo 205 pH meter (Testo Inc.,

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Sparta, NJ, USA), with a penetration electrode inserted directly into the muscle longissimus, in a section made at the 12th rib, according to the method of Beltran et al. (1997). The measurements

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were performed on all half-carcass at 2 hours and 24 hours post mortem. For glycogen, LT muscle samples between the 11th and 12th ribs (2 cm of diameter, ~20 g),

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collected only from the left half-carcass at 2 hours and 24 hours post mortem. Samples were immediately frozen in liquid nitrogen and placed in properly marked cryogenic tubes. Commercial enzymatic kits (Glycogen Assay Kit, Cayman chemical, MI, USA) were used for the determination of glycogen concentrations. Glycogen extraction was performed according to recommendations of the kit, where 350-400 mg of sample was weighed in 2 ml of buffer containing protease inhibitor (Assay Buffer). Then, samples were centrifuged at 800 x g of for 10 minutes at 4°C. The supernatant was transferred into another tube, which was diluted in 1:10 buffer solution and thereafter fluorescently read with excitation wavelength of 530-540 nm and 585-595 nm emission on SpectraMax M3 micro plate reader (Molecular Devices, LLC., CA, USA). 8

ACCEPTED MANUSCRIPT 2.5. Analyzed variables in meat Cooking loss was determined according to Wheeler et al. (2005). Steaks of 2.5 cm thickness were cooked in a grill preheated for 10 min, until reaching an internal temperature of 71ºC, which was controlled with thermocouples (Flyever Indústria e Comércio de Equipamentos Eletrônicos Ltda, São Carlos, São Paulo, Brazil) inserted into the geometric centre of each sample. The cooking loss was determined as the difference between initial and final weights, expressed as a percentage. From the cooked samples, cylindrical subsamples with known diameter (1.27 cm) cut parallel to the

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orientation of the fibres were extracted and later sheared using a Warner-Blatzler apparatus (TASBA CT3, Brookfield, USA) to determine shear force (Wheeler et al. 2005). The force required for shearing the samples was expressed in Newtons (N).

For the collagen analysis the methodology of the Near Infrared Spectroscopy (NIRS) was used by means of ISIscan - FoodScan ™ software. The meat color was determined by reading at

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three random points from the cutting surface of the LT muscle of each sample, using a portable spectrophotometer (CR-410-Konica Minolta, Camera Co., Ltd. Osaka, Japan) with D65 illuminant, aperture of 8 mm in diameter and observation angle of 10° (AMSA, 2012), previously calibrated with white standard, according to the manufacturer's instructions. The CIELAB system was used, taking into account light reflectance readings in three dimensions: L* (lightness), a* (redness) and

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b* (yellowness), according to a methodology described by Honikel (1998). The determination of the values for the hue angle (H*) was obtained according to MacDougal,

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(1994) and the determination of the oxymyoglobin and metamioglobin contents present on the surface of the meat (O/M) was performed according to Olivo and Shimokomaki ( 2001) using the coordinates of redness (a*) and yellowness (b*), obtained in the colorimetric determinations, using

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the following formulas: H* = tan-1 (b*/a*); O/M = (a*/b*). The ISIScan - FoodScan™ software was used to evaluate the centesimal composition using

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the Near Infrared Spectroscopy (NIRS) methodology. The methodology approved by the AOAC (2012), item 2007.04, involves freshly ground 180g in natura sample fit into a container for

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analysis, avoiding air bubbles, with optimum sample temperature of 10 - 20°C, and always making sure that the sample temperatures do not vary by more than 5°C. In this method, simultaneous determination of fat, moisture and protein of the meat were applied to the sample, with ranges of 1 43% or fat, 27 - 74% for moisture and 14 - 25% for protein. The altitude was 0.2000m; the temperature at 5 - 40°C; maximum relative humidity of 80% for temperatures of 0.31°C. The ashes were analyzed according to the method recommended by AOAC (2007), item 39.1.09. Cholesterol analysis was performed according to the enzymatic methodology described by Saldanha et al. (2004). Isolation of the lipids was carried out using 50% potassium hydroxide, ethyl alcohol and hexane. 3 mL of the hexane extract aliquot was dried under N2, and then isopropyl 9

ACCEPTED MANUSCRIPT alcohol was added until complete solubilization. Laborlab S/A laboratory kits, consisting of an enzymatic reagent (containing 0.025 mol/L 4-aminophenazone and 0.055 mol/L phenol) were used for quantification of cholesterol. 3 mL of working reagent were added to the samples and heat treatment was carried out for 10 minutes at 37°C in a water bath. After 90 minutes, the absorbance against the blank was read, also prepared at 499nm. The calibration curve was constructed on the basis of a standard cholesterol solution (1.006 mg/100 mL), with concentrations ranging from 0.01 to 0.05 mg/mL.

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Analysis of the fatty acid methyl esters was performed via gas chromatography using a Thermo 3300 chromatograph equipped with a flame ionization detector (FID) and a SEL-FAME CP-7420 fused silica capillary column (100 x 0.25 mm diameter x 0.25 mm thick). The temperature of the injector and detector was of 240°C. Column temperature was between 165°C and 235°C at 6°C min-1. The used gas flow rates were of 1.2 mL.min-1 for the carrier gas (H2), 30 mL.min-1 for

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the compensating gas (N2) and 35 and 350 mL.min-1 for the FID H2 gas and synthetic air, respectively. For the identification of fatty acids, retention times were compared with standard methyl esters (Sigma, USA), while quantification (mg of fatty acid per g of total lipids - TL) was performed against tricosanoic acid methyl ester (23:0) as the internal standard (IS), as described by Joseph and Ackman (1992). The theoretical values of the FID correction factor were used to obtain

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concentration values (Visentainer, 2012). The content of fatty acids was calculated using the following equation: FA = AXMISCFX/AISMXCFAE.

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In which FA is mg of TL fatty acid; AX is the peak area (fatty acids); AIS is the peak area of methyl ester of the IS tricosanoic acid (23:0); MIS is the weight (mg) of IS added to the sample; MX is the sample weight (mg); CFX is theoretical correction factor; CFAE is the conversion factor

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required to express the results as mg of fatty acid rather than methyl ester. The nutritional quality of the lipid fraction was evaluated by three indices from the fatty acid composition data, using the

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following calculations:

- Atherogenicity Index (IA) = [(C12:0 + (4 x C14:0) + C16:0)]/(ΣAGMI + Σω6 +Σω3);

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- Thrombogenicity Index (IT) = (C14:0 + C16:0 + C18:0)/[(0,5 x ΣAGMI) + (0,5 x Σω6 + (3 x Σω3) + (Σω3/Σω6)], according and Ulbricth e Southgate (1991); - The ratio between hypocholesterolemic and hypercholesterolemic fatty acids (HH) = (C18:1cis9 + C18:2ω6 + C20:4ω6 + C18:3ω3 + C20:5ω3 + C22:5ω3 + C22:6ω3)/(C14:0 + 16:0), according to Santos-Silva et al., (2002), where: AGMI = all monounsaturated acids. In addition, the ratios between AGI/AGS = Unsaturated / Saturated; AGP/AGS = Polyunsaturated / Saturated; ω6/ω3 = omega 6/omega 3, were measured. For the microbiological analysis, the Compedium of methods for the microbiological examination of foods (Downes and Ito, 2001) was taken as reference. Samples were prepared by 10

ACCEPTED MANUSCRIPT withdrawing aliquots (~10 g of each meat sample), which were then homogenized in 90 ml of 0.1% sterile peptone saline solution. Next, 1 mL of the first dilution (10-1) was transferred to a flask containing 9 mL of sterile peptone saline solution at 0.1% (10-2), and so on until reaching the dilution degree of 10-5. The total bacterial counts were followed as follows: 1 mL of each of the dilutions of each sample was deposited in sterile Petri dishes, then approximately 15 mL Standard Agar (PCA) was added for the total count of bacteria and Violet Crystal Agar Bile Dextrose (VBRD) for analysis of enterobacteriaceae, molten and cooled at around 45°C. The inoculum was

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mixed to the culture by means of gentle circular movements in the form of eight. After complete solidification of the medium, the plates were inverted and incubated at 32°C for 48 hours to determine total counts of bacteria and enterobacteriaceae and at 7°C for 10 days for the determination of psychrotrophic ones. For the colony count plates that contained between 25 and 250 colonies were selected. The counting was done with the aid of a magnifying glass coupled with

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a colony counter.

2.6. Statistical analysis

For the tests, 16 animals per treatment were used (4 animals per picket x 4 replicates per picket x 3 treatments), totaling 48 samples. The experimental design was arranged in complete,

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non-randomized blocks (block 1: pickets A, G and H, block 2: pickets B, I and J, block 3: pickets C, K and L, block 4: pickets D, E and F; Table 2), with three treatments and four replicates per

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treatment, totaling 12 experimental plots.

The data were analyzed using the MIXED procedure (SAS Institute Inc., Cary, NC, USA). We used the UNIVARIATE NORMAL procedure (SAS Inst. Inc., Cary, NC) and the data

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normality was confirmed by the Shapiro-Wilk test (W≥0.90). In the analysis of the data the animal was considered as an experimental unit for all variables studied. The data were then analyzed using

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the PROC MIXED procedure (SAS Institute Inc., Cary, NC, USA) and the Satterthwaite approximation to determine the degrees of freedom for the fixed effects tests. The system and the

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block were considered fixed effects, while the animal (picket) and the picket (treatment*block) were considered random effects. The means were calculated using the lsmeans procedure and the results were reported as square minimums and separated using the probability differences (pdiff) option, at the significance level of 5%. Kruskal-Wallis test was used for the non-parametric data (marbling score), with medians considered significantly different at P<0.05. The initial live weight of the termination phase was used as covariate to adjust the initial live weight of the termination phase and carcass characteristics. The trends were considered at P<0.09.

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ACCEPTED MANUSCRIPT 3. Results and Discussion The initial live weight presented a difference between the treatments (P = 0.01; Table 5), however, as the animals were in the area from the recreating phase, it was used as a covariate to adjust the carcass variables, in order to eliminate the effects of the system in the recreating phase and to evaluate only the termination phase. Thus, the final liveweight did not present differences between the evaluated treatments (P=0.77, Table 5) and, consequently, contributed to the absence of difference in hot carcass weight (P=0.54) and hot carcass yield P=0.94), forequarter weight

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(P=0,33) and forequarter yield (P=0.79), hindquarter weight (P=0,12) and hindquarter yield (P=0.93) with flank weight (P=0.96), and flank yield (P=0.28) between the ICL, ICLF-1L and ICLF-3L systems.

Among the carcass variables, the importance of warm carcass weight in Brazil's production systems is a consequence of the commercialization method used in the country, which remunerates

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the producer in accordance with this characteristic (Lopes et al., 2012). Therefore, it can be stated that economically speaking, the animals presented similar carcasses in the different integration systems, since there was no difference in the warm carcass weights among the evaluated treatments. In addition, satisfactory carcass yield was obtained in all treatments from a productive point of view, considering that many slaughterhouses conform to carcass yield of just 50% when they buy

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the animal based on their live weight (Lopes et al. 2012).

The absence of difference between various treatments in weight and yield of the hindquarter

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of the animal, where the noblest cuts of the carcass are to be found, also indicates that they had the same degree of finish, an important fact for the productive system. Advantages, especially, for the slaughterhouses industry, due to its noble courts, in this case, with no differences in market prices

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(Missio et al., 2010).

No difference was found between treatments for the values initial ribeye area (P=0.70) and

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final ribeye area (P=0.86), initial subcutaneous fat thickness (P=0.77) and final subcutaneous fat thickness (P=0.69), as well as marbling score (P=0.49; Table 5). The ribeye area has a high

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relationship with growth rate, increasing as the animal increases its meat deposition in the carcass (Lopes et al., 2012). This explains the absence of differences between the ribeye area of the valuated animals, because there was no difference in the carcass parameters between the animals submitted to the different integration systems. The microclimate provided by the arboreal component in its different densities did not interfere with the subcutaneous fat thickness of the animals, indicating that the Nellore cattle had a higher tolerance to heat. In general, when animals are submitted to high temperature environments, thermoregulatory mechanisms are activated to reduce the impact of the warm environment on their organism, which causes some of the net energy used for the deposition of these tissues to be lost 12

ACCEPTED MANUSCRIPT (Orlando et al., 2001). On the other hand, fat deposition can be compensated by the internal deposits and also can favor the marbling (Mader and Davis, 2004; Nardone et al., 2006), which deposits constantly in the animal, but is only expressed during later stages (Cianzio et al., 1982; Pethick, et al., 2004). This explains the absence of differences in the marbling between the treatments, since there was also no difference in the subcutaneous fat thickness of the carcass of the animals between the different integration systems. Furthermore, the final subcutaneous fat thickness found in all treatments presented mean

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values higher than the minimum required by the refrigeration industry (3 mm) and also with values able to protect the carcass against darkening, dehydration and shortening of the fibers due to cold during the cooling process (EGS> 5 mm), thus contributing to the visual appearance of the carcass and to the quality of the meat and ensuring its softness (Felício, 1997). The marbling was classified in all treatments as being between mild and low (USDA, 1997), which is characteristic of zebu lean

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meat, which may be an advantage for human health (Climaco et al., 2011).

The pH of the carcasses (P = 0.33 and P = 0.62), as well as muscle glycogen (P = 0.99 and P = 0.97) at 2h and 24h after slaughter, respectively, did not show differences between treatments (Figure 3), reinforcing the hypothesis that the natural darkening did not influence the carcass characteristics of Nellore cattle. It can be also stated that glycogen presented adequate levels

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(152.95 μg/ml, 153.03 μg/ml and 155.15 μg/ml at 2 h post-mortem and 47.05 μg/ml, 41.85 μg/ml and 45.33 μg/ml at 24 h post-mortem for ICL, ICLF-1L and ICLF-3L, respectively) for a decrease

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in muscle pH (6.96, 6.82 and 7.00 at 2 h post mortem and 5, 52, 5.53 and 5.51 at 24 h post-mortem for ICL, ICLF-1L and ICLF-3L, respectively). The normal pH should be between 5.5 and 5.7 24 h post mortem (Kandeepan and Biswas, 2007) to ensure the normal patterns of development of the

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physical and chemical characteristics responsible for meat quality. In addition, the final pH of the longissimus thoracis muscle in all evaluated treatments (Table 6) was lower than the pH limit

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defined for the export of meat to the European Union (pH = 5.9, according to Circular 192/98/DCI/DIPOA). Thus, had the animals presented caloric stress due to exposure to the sun, this

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glycogen would be almost completely depleted in the muscle, not producing enough lactic acid for the pH drop (Apple et al., 2006). However, this fact does not prevent the recommendation of shading for Nellore cattle, since this system allows better quality of life for these animals (Ferro et al., 2016). Thus, the absence of difference for the parameters of weight losses by digesting (P = 0.37), shear strength (P = 0.54), collagen (P = 0.44) and meat color (P = 0.29, P = 0.15, P = 0.57 for a*, b* and L*, respectively) between treatments was expected (Table 6), and since no biochemical changes were observed during the post mortem period, there were no changes in color, 13

ACCEPTED MANUSCRIPT appearance, taste, texture (softness and juiciness) and functional properties of meat (Ramos and Gomide 2007). Among the parameters of meat quality, tenderness is the characteristic, which is prized the most by the consumer, and consumer satisfaction in Latin American countries is achieved when the shear strength is equal to or less than 40.13 N (Rodas-González et al., 2009). However, the meat of the animals of the present study presented values higher than the desired ones, possibly because they were animals fed on pasture, reaching slaughter weight at a more advanced age (34 months)

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(Parmigiani and Torres, 2009). In addition, the meat of zebu animals presents a difference in the action of proteolytic enzymes during the post-slaughter period (Curi et al., 2009), considered less tender due to the higher activity of calpastatin, which inhibits calpain, (Hadlich et al., 2008). The color attribute, which represents the first impact on the consumer, showed luminosity (L*) and intensity of red (a*) values within the proposed standards for beef, ranging from 33.2-41.0

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and between 11.1-23.6, respectively (Muchenje et al., 2009). On the other hand, the means for the intensity of yellow (b*) were considered low (Abularach et al., 1998, Muchenje et al., 2009). However, this reduction in the mean values of b* did not cause differences in the hue angle (H*, P = 0.80, Table 6), which is directly related to a* and b*. There is no standard values for this parameter, though. It is suggested that the increase in H* values is usually accompanied by the meat

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discoloration process (Lee et al., 2005). The absence of discoloration can be further confirmed by the amount of oxymioglobin in relation to metamioglobin (O/M) present on the surface of the meat,

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which showed no differences between the treatments (P = 0.54; Table 6), however, the higher values of this variable in the results indicate a higher concentration of oxymyoglobin, which is responsible for giving a bright red color to the meat, while values close to zero indicate higher

The

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concentrations of metamioglobin (Olivo and Shimokomaki, 2001). different

integration

systems

did

not

interfere

with

the

protein

content

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(P = 0.99), ethereal extract (P = 0.22), moisture (P = 0.97), ashes (P = 0.44) and cholesterol (P = 0.21) of the Nellore cattle meat (Table 7). According to Geay et al. (2001) the amount of protein,

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moisture and ashes of meat is generally constant, with approximately 75% water, 19% to 25% protein and 1% to 2% minerals, which means that the flesh of animals of the present study was in accordance with the ranges suggested in the literature. On the other hand, the nature and quantity of lipids vary significantly, depending, among other factors, on external conditions, such as the type of feed offered (Geay et al., 2001). In addition, when the animal is exposed to high temperatures for long periods, it can affect the composition of the carcass and meat by reducing the deposition of subcutaneous fat and greater deposition of fat in the internal deposits (Gregory, 2010). In this concept, the absence of difference between the ethereal extract and cholesterol variables, which present a significant correlation as a function of ethereal extract contents, includes 14

ACCEPTED MANUSCRIPT the intercellular and intracellular fat, the latter of which contains the highest cholesterol levels. (Stromer et al. 1966; Arboitte et al., 2004), suggest that, although there are changes in the bromatological composition of the grasses depending on the tree component, apart from the fact that the shade of the trees provides microclimate favorable to Nellore animals (Tables 1 and 3), it does not affect the composition of the meat of the same. However, there was a tendency for cis-10-heptadecenoic (C17:1n9) and arachidonic (C20:4n6) fatty acids (P = 0.05 and P = 0.06, respectively; Table 8), both showing higher values in

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the meats of the animals submitted to ICL and ICLF-1L treatments. However, this trend was not able to change the amount of total PUFAs (P = 0.49) or the ω6: ω3 ratio (P = 0.91; Table 9), which did not show any difference between treatments.

In fact, among the polyunsaturated fatty acids of the omega 6 series, arachidonic is one of the most representative and plays an important role in the inflammatory process and in a series of

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physiological systems, such as the renal, gastrointestinal, reproductive and cardiovascular systems, apart from being a mediator in immune and inflammatory responses (Curi et al., 2002). However, in order to promote the beneficial action on human health in the prevention of cardiovascular diseases, atherosclerosis and chronic inflammatory diseases, as well as due to its anti-inflammatory and antithrombotic action, it is important to maintain adequate balance between these and omega-3 fatty

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acids (Andrade and Carmo, 2006, Lavie et al., 2009, Mickleborough, 2009). Epidemiological studies and the Japanese Society of Lipid Nutrition have reported that the

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ω6:ω3 ratio recommended as an ideal for daily human intake is 4: 1 for healthy adults (Department of Health and Social Security, 1994; Uauy et al., 1999). The meat of the animals of the present study presented values lower than three, considered, therefore, healthy for human consumption in

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all evaluated treatments. This result was expected, since the animals came from a predominantly

3).

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pasture-based regime, which contains a greater amount of α-linoleic (C18:3n3) in the grasses (Table

The nutritional quality index (NQI) did not show any difference between the treatments

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(Table 9). However, there was a tendency for a higher thrombogenicity index value for the ICLF-3L treatment (IT; P = 0.08; Table 9). Among the IQN variables, the AGP/AGS ratio in all evaluated treatments was below that

recommended for human diets, which should be around 0.45 in order to prevent cardiovascular problems (Department of Health and Social Security, 1994). In general, the AGP/ AGS ratio is lower in ruminant meat due to dietary biohydrogenation by rumen microorganisms (French et al., 2000). Calculation of the HH fatty acid ratio, more specifically related to blood cholesterol metabolism, resulted in values in the range of 1.02 to 1.22, high values are desirable for meat

15

ACCEPTED MANUSCRIPT products in the nutritional sense, as these are able to reduce the risk of cardiovascular diseases (Santos-Silva et al., 2002). In contrast, for IA and IT indices, it is considered that the lower the value, the more favorable is the profile of fatty acids to human health (Sousa Bentes et al., 2009). The IA, which correlates with the pro and antiatherogenic acids in the longissimus thoracis muscle of the tested animals, was between 0.92 and 1.04, which is above the recommended ideal level (IA = a maximum of 0.72; Ulbricht and Southgate, 1991). In relation to IT, although there was a trend of higher values in the

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meat of the animals in ICLF-3L systems, all treatments also presented a level higher than the recommended limit (IT = a maximum of 1.27, Ulbricht and Southgate, 1991), thus indicating , that the trend is not justified by the use of trees in the system, since the undesirable result is found in all treatments.

Regarding the microbiological quality of the Longissimus thoracis muscle, no difference was

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observed for the total bacterial count (P = 0.48), group of psychrotrophic bacteria (P = 0.46) and enterobacteria (P = 0.42) between treatments (Table 10). In addition, the discovered microbial load does not classify the meat according to the degree of deterioration, since, in order to be considered contaminated, the meat must have levels above 6 or 7 log UFC / g for these groups of bacteria (Fung et al. 1980; Capta et al., 1999). This result was expected, since the pH, which, when showing

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high values, is responsible for the increase of the microbial load in the meat (Borges and Freitas,

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2002), showed average values, suitable for meat maintenance (5.4 – 5.8) (Mach et al., 2008).

4. Conclusions

The introduction of the tree component in the pasture, in its different densities, does not

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interfere in the characteristics of the carcass, in the composition and the physical and microbiological quality of the Nellore cattle meat when compared to the integration system without

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trees, thus showing that this cattle are adapted to climatic conditions of the region to the point of the latter not affecting the meat. Therefore, from the point of view of carcass and meat quality,

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integrated crop-livestock systems with and without the presence of trees can be recommended for Nellore cattle.

5. Acknowledgment APTA, Paulista Agency for Agribusiness Technology, Polo Regional Extreme West Andradina/SP for logistical, infrastructural support and assistance during the experiment. To the Research Foundation of the State of São Paulo - FAPESP (2014/12662-0) for the scholarship. The opinions, hypotheses, conclusions or recommendations contained in this material are the responsibility of the authors and do not necessarily reflect the views of FAPESP. 16

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M., Folke, C., Schellnhuber, H.J., Nykvist, B., Wit, C.A., Hughes, T., van der Leeuw, S., Rodhe, H., Sörlin, S., Snyder, P.K., Costanza, R., Svedin, U., Falkenmark, M., Karlberg, L., Corell, R.W., Fabry, V.J., Hansen, J., Walker, B., Liverman, D., Richardson, K., Crutzen, P., Foley, J.A. 2009.

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A safe operating space for humanity. Nature. 461: 472–475. doi:10.1038/461472a. Rodas-González, A., Huerta-Leidenz, N., Jerez-Timaure, N., Miller, M.F.

2009. Establishing

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tenderness thresholds of Venezuelan beef steaks using consumer and trained sensory panels. Meat Sci. 83: 218-223. doi: 10.1016/j.meatsci.2009.04.021.

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Saldanha, T., Mazalli, M.R., Bragagnolo, N. 2004. Avaliação comparativa entre dois métodos para determinação do colesterol em carnes e leite. Ciênc. Tecnol. Aliment. 24: 109-113. doi: 10.1590/S0101-20612004000100020.

Sánchez, M.D. 2001. Panorama dos sistemas agroflorestais pecuários na América Latina. In: Carvalho, M.M., Alvim, M.J., Carneiro, J.C. (Eds). Sistemas agroflorestais pecuários: opções de sustentabilidade para áreas tropicais e subtropicais. Juiz de Fora: Embrapa Gado de Leite, 9-17. (In Portuguese).

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ACCEPTED MANUSCRIPT Santos-Silva, J., Bessa, R.J.B., Santos-Silva, F. 2002. Effect of genotype, feeding system and slaughter weight on the quality of light lambs. II. Fatty acid composition of meat. Livest. Prod. Sci. 77: 187-194. doi: 10.1016/S0301-6226(02)00059-3. SAS Institute. 2010. SAS User’s guide: Statistics. Version 9.3. Cary, NC. Silva, J.A.R.; Araújo, A.A.; Lourenço Júnior, J.B., Santos, N.F.A., Garcia, A.R., Nahúm, B.S. 2011. Conforto térmico de búfalas em sistema silvipastoril na Amazônia Oriental. Pesq. Agropec. Bras. 46: 1364-1371. doi: 10.1590/S0100-204X2011001000033. Silva, S.C. 2004. Fundamentos para o manejo do pastejo de plantas forrageiras dos gêneros Brachiaria e

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Panicum. In: Simpósio Sobre Manejo Estratégico da Pastagem, 2, 2004, Viçosa. Anais. Viçosa: UFV, 347-385. (In Portuguese).

Sousa Bentes, A., Souza, H.A.L., Simões, M.G., Mendonça, X.M.F., 2009. Caracterização física e química e perfil lipídico de três espécies de peixes amazônicos. RBTA. 3: 97-108. doi: 10.3895/S1981-36862009000200011.

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Stromer, M.H., Goll, D.E., Roberts, J.H. 1966. Cholesterol in subcutaneous and intramuscular lipid depots from bovine carcasses of different maturity and fatness. J Anim Sci. 25: 1145-1152. doi: 10.2527/jas1966.2541145x.

Uauy, R., Mena, P., Valenzuela, A. 1999. Essential fatty acids as determinants of lipids requeriments in infants, children and adults. Eur. J. Clin. Nutr. 53: 66-67.

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Ulbrich, T.L.V. Southgate, D.A.T. 1991.Coronary heart disease: seven dietary factors. Lancet, 338(8773): 985-992. doi: 10.1016/0140-6736(91)91846-M.

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Unicamp - Universidade Estadual de Campinas 2012. Centro de Pesquisas Meteorológicas e Climáticas Aplicadas a Agricultura. Clima dos Municípios Paulistas. Andradina. Available at: http://www.cpa.unicamp.br/outras-informacoes/clima_muni_024.html (accessed March 30, 2017).

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(In Portuguese).

USDA/ARS. 1997. US Department of Agriculture, Agricultural Research Service. Nutrient Data

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Laboratory. Search The USDA National Nutrient Data base for standard national nutrient data base for Standard Reference, Release 18.

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van Raij, B., Cantarella, H., Quaggio, J.A., Furlani, A.M.C. (Ed.). Recomendações de adubação e calagem para o Estado de São Paulo. 2.ed. rev. e atual. Campinas: Instituto Agronômico/Fundação IAC, 1997. 285p. (In Portuguese).

Visentainer, J.V. 2012. Aspectos analíticos da resposta do detector de ionização em chama para ésteres de ácidos graxos em biodiesel e alimentos. Quím. Nova. 35: 274-279. doi: 10.1590/S010040422012000200008. Wirsenius, S., Azar, C., Berndes, G., 2010. How much land is needed for global food production under scenarios of dietary chances and livestock productivity increases in 2030? Agric. Sys. 103: 621638. doi: 10.1016/j.agsy.2010.07.005. 22

ACCEPTED MANUSCRIPT Wood, J.D., Enser, M. Fisher, A.V., Nute, G.R., Richardson, R.I., Sheard, P.R. 1999. Manipulating meat

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quality and composition. Proc. Nutr. Soc. 58: 363-370. doi: 10.1017/S0029665199000488.

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ACCEPTED MANUSCRIPT Table 1 Rainfall and maximum and minimum temperatures during the experimental period and the last 50 years in Andradina - São Paulo and descriptive analysis of climatic conditions and thermal comfort of the experimental area. Jan.

Fev.

Mar.

Abr.

Monthly rain (mm) Mean max. temp. (ºC) Mean min. temp. (ºC)

66.2 35.4 21.6

141.6 33.0 16.6

194.0 32.1 16.5

42.6 34.2 11.2

Monthly rain (mm) Mean max. temp. (ºC) Mean min. temp. (ºC)

372.4 31.7 21.5

102.2 33.5 21.6

74.8 32.2 20.3

61.0 34.9 19.9

Monthly rain (mm) Mean max. temp. (ºC) Mean min. temp. (ºC)

210.5 31.7 20.1

166.0 31.9 20.3

129.7 31.7 19.7

69.7 30.6 17.1

Climate characteristics2

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ICL Wind speed (m/s) 1.09 ± 0.61 Dry Bulb Temp (ºC) 33.23 ± 5.43 Relative humidity (%) 45.83 ± 13.85 423.69 ± 122.29 Radiant Heat Charge (W.m-2) Temp and Humidity Index 83.92 ± 5.14 Globe Temp and Humidity Index 87.22 ± 7.53 * Experimental period: January to June 2016. 1 Unicamp (2013). 2 Data collected in the experimental area.

Months Jun. Jul. Ago. Set. Out. Nov. Dez. 2015 36.6 21.3 44.1 11.4 115.0 160.4 203.9 61.2 28.8 27.0 26.6 32.1 32.3 34.2 31.9 32.1 16.5 15.6 15.8 25.3 18.4 19.8 20.8 21.0 2016* 136.4 51.6 5.0 67.0 43.6 89.8 51.6 149.6 28.0 33.5 29.9 31.6 30.0 32.9 32.8 34.4 16.2 2.6 13.4 14.9 15.8 19.1 20.0 20.6 Long-term (50 yr) avg. 59.2 31.9 23.2 22.5 57.5 114.5 121.9 175.0 28.8 27.8 28.1 30.8 31.9 32.0 31.9 31.5 14.5 13.3 12.7 14.4 16.5 18.1 18.7 19.7 Treatment ICLF-1L ICLF-3L Max. Min. 0.80 ± 0.54 0.59 ± 0.34 1.90 0.03 32.14 ± 5.65 32.12 ± 5.72 39.17 22.94 49.60 ± 17.48 51.37 ± 16.24 80.99 34.27 530.52 ± 45.96 523.13 ± 96.44 257.98 735.37 81.33 ± 5.64 81.75 ± 6.00 90.53 71.80 86.07 ± 6.48 84.55 ± 14.31 110.22 42.60 Mai.

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Climate characteristics1

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ACCEPTED MANUSCRIPT Table 2 Picket and experimental area treatments, Andradina – São Paulo, Brazil.

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Picket Area (ha) Production system 1,88 Integration crop-livestock (ICL) A 1,95 Integration crop-livestock (ICL) B 1,81 Integration crop-livestock (ICL) C 2,04 Integration crop-livestock (ICL) D 2,42 Integration crop-livestock-forest, with trees in triple lines (ICLF-3L) E 1,85 Integration crop-livestock-forest, with trees in single lines (ICLF-1L) F 2,21 Integration crop-livestock-forest, with trees in single lines (ICLF-1L) G 1,97 Integration crop-livestock-forest, with trees in triple lines (ICLF-3L) H 2,14 Integration crop-livestock-forest, with trees in single lines (ICLF-1L) I 1,79 Integration crop-livestock-forest, with trees in triple lines (ICLF-3L) J 1,95 Integration crop-livestock-forest, with trees in single lines (ICLF-1L) K 1,65 Integration crop-livestock-forest, with trees in triple lines (ICLF-3L) L 23,66 Total ICLF-1L: Eucalyptus trees planted in single lines, the distance between each eucalyptus range being 17 m to 21 m and the distance between plants of 2 m, with a density of 196 trees.ha -1; ICLF-3L: Eucalyptus trees planted in triple lines, the distance between the eucalyptus ranges being 17 m to 21 m, the distance between plants of 2 m and the distance between eucalyptus lines of 3 m, with a density of 448 trees.ha-1.

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ACCEPTED MANUSCRIPT Table 3 Descriptive analysis of the production and chemical composition of Urochloa brizantha, cv. Marandu during the experimental period. Pasture characteristics

ICL 43.85 ± 12.94 5,400 ± 945.28 27.00 ± 4.36 16.78 ± 2.80 56.25 ± 6.36

Accumulation rate (kg of DM/ha.day) Forage mass (kg/ha) % Stem % Leaf % Dead material Chemical composition

Max. 66.25 6.443 35.50 21.25 65.75 Max. 37.39 11.23 16.96 74.24 36.75 41.05 31.58 5.60

Min. 14.05

2.614 20.50 11.50 43.75

Min. 17.79 6.24 7.83 46.46 22.46 24.00 19.41 2.50

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ICL 25.69 ± 0.34 9.25 ± 0.15 10.55 ± 0.26 68.85 ± 0.42 33.81 ± 0.25 35.04 ± 0.35 29.59 ± 0.37 4.43 ± 0.13

Dry matter (%) Mineral Matter (%) Crude Protein (%) NDF (%) FDA (%) Hemicellulose (%) Cellulose (%) Lignin (%)

Treatment ICLF-1L ICLF-3L 38.97 ± 19.72 38.78 ± 11.57 4,161 ± 833.26 4,250 ± 958.27 29.56 ± 4.17 29.26 ± 4.66 18.72 ± 3.36 18.18 ± 2.35 51.78 ± 7.06 52.45 ± 6.50 Treatment ICLF-1L ICLF-3L 22.91 ± 0.37 22.80 ± 0.36 9.14 ± 0.15 8.98 ± 0.15 12.55 ± 0.26 12.44 ± 0.26 66.98 ± 0.42 67.46 ± 0.51 33.28 ± 0.25 33.43 ± 0.26 33.70 ± 0.35 34.25 ± 0.36 29.17 ± 0.37 28.99 ± 0.57 4.11 ± 0.13 4.34 ± 0.13

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ACCEPTED MANUSCRIPT Table 4 Ingredients and nutritional composition of the supplement offered to the animals between April and June 2016.

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Ingredients, % of DM Ground corn 82.21 Soybean meal 11.86 Urea 1.11 Mineral Supplement* 4.91 1 Chemical composition , DM basis DM, % as fed basis 87.93 2 ME , Mcal/kg 3.10 NEm2, Mcal/kg 2.11 2 NEg , Mcal/kg 1.44 CP, % 16.90 MP2, % 15.02 NDF, % 10.21 3 peNDF , % 3.20 NFC3, % 64.97 EE3, % 4.11 Ca, % 0.50 P, % 0.27 *Levels of minimum guarantee per kg of product: Crude protein (min.) = 210.0000 g/kg; NNP-Equiv. protein (max.) = 150,000 g/kg; Estimated NDT = 280.0000 g/kg; Calcium (min.) = 65.0000 g/kg; Calcium (max) = 85.0000 g/kg; Phosphorus (min) = 30.0000 g/kg; Sodium (min) = 100.0000 g/kg; Magnesium (min) = 10.0000 g/kg; Sulfur (min) = 15.0000 g/kg; Cobalt (min) = 100.0000 mg/kg; Copper (min) = 800.0000 mg/kg; Iodine (min) = 100.0000 mg/kg; Manganese (min) = 1,500,0000 mg/kg; Selenium (min) = 20,0000 mg/kg; Zinc (min) = 3.200.0000 mg/kg; Iron (min) = 1,500,0000 mg/kg; Fluorine (max) = 300.0000 mg/kg; Bacillus subtilis (Min.) = 4.5000x109 UFC/kg; Bifidobacterium bifidum (min.) = 1.5000x109 CFU/kg; Enterococcus faecium (min.) = 1.5000x109 CFU/kg; Lactobacillus acidophilus (min.) = 1.5000x109 CFU/kg; Lactobacillus buchneri (min.) = 3.0000x109 UFC/kg; Lactobacillus casei (min.) = 1.5000x109 CFU/kg; Lactobacillus lactis (min.) = 1.5000x109 CFU/kg; Saccharomyces cerevisiae (min.) = 1.0000x109 CFU/kg. 1 DM = Dry matter; ME = Metabolizable energy; NEm = net energy for maintenance; NEg = net energy for gain; CP = crude protein; MP = metabolizable protein; NDF = neutral detergent fiber; peNDF = physically effective neutral detergent fiber; NFC = non-fibrous carbohydrate and EE = ethereal extract; 2 Values calculated using the NRC (2001).

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Table 5 Initial liveweight (ILW), final liveweight (FLW), hot carcass weight (HCW), carcass yield (CY), forequarter weight (FQW), forequarter yield (FQY), hindquarter weight (HQW), hindquarter yield (HQY), flank weight (FW), flank yield (FY), initial ribeye area (REA-i), final ribeye area (REA-f), initial subcutaneous fat thickness (SFT-i), final subcutaneous fat thic (SFT-f) and marbling (MAR) of Nellore cattle in integration crop-livestock (ICL), integration crop-livestock-forest with density of 196 ha-1 trees (ICLF-1L) and integration crop-livestock-forest with density 448 ha-1 trees (ICLF-3L). HQW % 47.79 48.63 48.57

FW kg 17.02 16.12 16.51

Standard error 4.728 5.396 2.944 0.212 0.605 0.139 0.661 0.133 § 0.017 0.772 0.549 0.948 0.333 0.797 0.121 0.937 P-value Data submitted to a covariance of initial live weight use for final live weight and carcass variables. § Different letters in the same column differ from each other by Student t (P<0.05). *Non-parametric Kruskal-Wallis test, the medians considered to be significantly different when P<0.05.

0.247 0.962

FLW kg 443 448 455

HCW kg 255.16 256.98 258.66

CY % 57.48 57.40 56.82

FQW kg 50.22 52.41 55.22

FQY % 39.03 39.29 39.10

HQW kg 63.87 64.84 68.53

FY % 13.17 12.27 12.32

REA-i

REA-f cm2 52.48 75.19 52.75 75.24 52.95 76.92

SFT-i

SFT-f mm 1.80 5.85 1.69 5.93 1.90 6.05

MAR* 1-10 3.30 3.43 3.93

0.109 0.286

0.709 0.341

0.084 0.777

0.094 0.490

1.739 0.866

0.353 0.697

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ICL ICLF-1L ICLF-3L

ILW kg 400 a 373 b 382 b

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Decline of glycogen post mortem

Decline of pH post mortem

Glycogen (µg/ml)

8 7 5 4 3 2

1 0 ICLF-1L pH 2h

ICLF-3L

180 160 140 120 100 80 60 40 20 0

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ICL

Glycogen 2h

pH 24h

ICLF-1L

ICLF-3L

Glycogen 24h

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Figure 1. Hydrogen potential (pH) and glycogen concentration (µg/ml) of longissimus thoracis muscle at 2 h e 24 h post mortem of Nellore cattle in integration crop-livestock (ICL), integration crop-livestock-forest with density of 196 ha-1 trees (ICLF-1L) and integration crop-livestock-forest with density 448 ha-1 trees (ICLF-3L), (P = 0.33, EP = 0.05; P = 0.62, EP = 0.01; P = 0.99, EP = 15.20; P = 0.97; EP = 6.96 for pH 2 h, pH 24 h, glycogen 2 h and glycogen 24 h, respectively).

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Table 6 Hydrogen potential (pH), Cooking loss (CL), shear force (SF), collagen, lightness (L*), redness (a*), yellowness (b*), hue angle (H*) e oxymyoglobin and metamioglobin (O/M) of longissimus thoracis muscle of Nellore cattle in integration crop-livestock (ICL), integration crop-livestock-forest with density of 196 ha-1 trees (ICLF-1L) and integration crop-livestock-forest with density 448 ha-1 trees (ICLF-3L). pH

ICL ICLF-1L ICLF-3L

5.52 5.53 5.51

CL % 24.01 25.50 24.90

SF N 61.09 65.41 64.42

Collagen % 1.30 1.35 1.35

L*

a*

b*

H*

O/M

38.34 38.84 39.08

14.92 14.93 15.50

2.48 2.66 2.72

9.48 9.68 9.82

6.57 6.03 6.14

0.176 0.290

0.142 0.153

0.091 0.579

0.318 0.806

0.209 0.545

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Standard error 0.008 0.429 0.166 0.014 § 0.625 0.373 0.548 0.442 P-value § Different letters in the same column differ from each other by Student t (P<0.05).

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Moisture % 73.09 73.10 73.20

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Ashes % 1.11 1.12 1.10

Colesterol mg 100g of meat-1 48.39 52.47 52.01

0.139 0.974

0.006 0.440

1.099 0.211

Treatment

Protein % 23.45 23.57 23.42

ICL ICLF-1L ICLF-3L

Fat % 2.42 2.30 2.39

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Standard error 0.093 0.090 § 0.991 0.226 P-value § Different letters in the same column differ from each other by Student t (P<0.05).

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Table 7 Protein, fat, moisture, ashes and cholesterol of longissimus thoracis muscle of Nellore cattle in integration crop-livestock (ICL), integration croplivestock-forest with density of 196 ha-1 trees (ICLF-1L) and integration crop-livestock-forest with density 448 ha-1 trees (ICLF-3L).

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ACCEPTED MANUSCRIPT Table 8 Fatty acid profile of longissimus thoracis muscle of Nellore cattle in integration crop-livestock (ICL), integration crop-livestock-forest with density of 196 ha-1 trees (ICLF-1L) and integration crop-livestock-forest with density 448 ha-1 trees (ICLF-3L). Treatment § Standard error P-value ICLF-1L ICLF-3L mg of fatty acid g of total fat-1 C12:0 0.05 0.05 0.06 0.004 0.498 C14:0 2.04 1.95 2.04 0.187 0.976 C14:1n9 0.58 0.51 0.56 0.058 0.888 C14:1n7 0.33 0.32 0.45 0.035 0.299 C15:0 0.44 0.45 0.61 0.046 0.226 C15:1n5 0.23 0.23 0.28 0.023 0.543 C16:0 17.77 16.23 16.88 1.476 0.929 C16:1n9 0.18 0.17 0.19 0.015 0.898 C16:1n7 0.88 0.89 0.98 0.085 0.879 C16:1n5 0.41 0.40 0.41 0.036 0.987 C17:0 1.03 1.06 1.21 0.098 0.728 C17:1n9 0.15 a 0.15 a 0.12 b 0.008 0.055 C18:0 16.65 16.57 19.84 1.439 0.546 C18:1trans11 1.33 1.28 1.79 0.135 0.218 C18:1n9c 20.87 19.25 18.17 1.777 0.855 C18:1n7 0.26 0.30 0.27 0.025 0.765 C18:2n6 0.92 0.97 0.80 0.039 0.429 C18:3n6 0.09 0.09 0.09 0.009 0.962 C18:3n3 0.27 0.27 0.24 0.010 0.359 C20:2n6 0.07 0.06 0.05 0.003 0.306 C20:3n6 0.07 0.06 0.05 0.003 0.217 C20:4n6 0.19 a 0.19 a 0.16 b 0.007 0.063 C20:5n3 (EPA) 0.05 0.06 0.04 0.003 0.171 C22:5n3 (DPA) 0.14 0.14 0.11 0.006 0.172 C22:6n3 (DHA) 0.02 0.02 0.01 0.001 0.117 C23:0 5.06 5.10 5.08 0.011 0.603 SFAs 37.99 36.30 40.65 3.139 0.856 MUFAs 25.78 23.97 23.23 2.198 0.909 PUFAs 2.21 2.26 1.96 0.085 0.498 Omega-6 1.36 1.42 1.16 0.056 0.348 Omega-3 0.49 0.48 0.40 0.018 0.223 CLA 0.36 0.36 0.39 0.037 0.918 EPA, icosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid § Different letters in the same column differ from each other by Student t (P<0.05). Fatty acid profile

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Tablea 9 Ratio between unsaturated and saturated fatty acids (UFAs/SFAs), polyunsaturated and saturated (PUFAs/SFAs), omega 6 and omega 3 (ω6/ω3) hypocholesterolemic and hypercholesterolemic (HH), atherogenicity index (AI) and thrombogenicity index (TI) of fat of longissimus thoracis muscle of Nellore cattle in integration crop-livestock (ICL), integration crop-livestock-forest with density of 196 ha-1 trees (ICLF-1L) and integration croplivestock-forest with density 448 ha-1 trees (ICLF-3L). UFAs/SFAs*

PUFAs/SFAs

ICL ICLF-1L ICLF-3L

0.74 0.72 0.62

0.06 0.05 0.05

HH mg of fatty acid g of total fat-1 2.96:1 1.17 2.98:1 1.22 2.86:1 1.02

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2.33 a 2.32 a 2.84 b

0.105 0.915

0.028 0.231

0.085 0.088

0.037 0.175

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Standard error 0.035 0.002 § 0.223 0.531 P-value * UFAs = monounsaturated + polyunsaturated.. § Different letters in the same column differ from each other by Student t (P<0.05).

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Table 10 Total bacterial count (TBC), psychrotrophic (PSY) and enterobacteriaceae (ENT) of longissimus thoracis muscle of Nellore cattle in integration croplivestock (ICL), integration crop-livestock-forest with density of 196 ha-1 trees (ICLF-1L) and integration crop-livestock-forest with density 448 ha-1 trees (ICLF-3L). TBC

ICL ICLF-1L ICLF-3L

0.66 0.81 0.79

ENT

0.048 0.467

0.056 0.429

0.54 0.70 0.61

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Standard error 0.052 § 0.486 P-value § Different letters in the same column differ from each other by Student t (P<0.05).

PSY CFU log g-1 0.86 0.98 0.88

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